α-helical membrane proteins (HMPs) comprise the largest subfamily of integral membrane proteins which make up for about 30% of the eukaryotic genome [1]. HMPs proteins exhibit one or more α-helices (also called transmembrane helices or TM helices) that span the cellular membrane and play important roles in a variety of cellular processes like signal transduction, transport of ions or molecules across membranes, metabolism, cell-cell communication. Some of the members include bacteriorhodopsin, cytochrome c oxidases, voltage-gated ion channels, aquaporins, and G protein-coupled receptors (GPCRs).
The HMP family is dominated by proteins with 7 TM α-helices called 7TM proteins. The GPCRs which also contain 7 TM α-helices makeup for the majority of the 7TM proteins, as a small number of 7TM proteins do not couple to G proteins. The 7TM proteins are activated by a variety of bioactive molecules, like biogenic amines, peptides, lipids, nucleotides, proteins, tastants, odorants and non-molecular sensory signals like light, touch etc [2], making them essential for a range of physiological processes (e.g. neurotransmission, cellular metabolism, secretion, cell growth, immune defense, and differentiation). They are also the largest family in human genome with about 800 7TM proteins identified, including about more than 400 non-sensory receptors organized into 5 families (GRAFS): Glutamate, Rhodopsin, Adhesion, Frizzled/Taste2, and Secretin [3]. Due to their mediation of numerous critical physiological functions, 7TM proteins are implicated in all major disease areas including cardiovascular, metabolic, neurodegenerative, psychiatric, cancer and infectious diseases. Indeed these proteins represent 30-50% of the current drug targets for activation (by agonists) or inhibition (by antagonists).
Other members of the HMP family are also involved in a range of critical functions like photosynthesis, proton transport (Bacteriorhodopsin), electron transfer in enzymatic reactions (Cytochrome c oxidase), transport of critical substrates across membranes (ABC transporters), translocation of other membrane proteins during/after their synthesis (SecY), voltage-gated ion channels (potassium channels), and water transport across membranes (aquaporins) to name a few.
Biochemical and biophysical studies of HMPs have accelerated progress in identifying various subtypes with specific cell and tissue functions. However, progress in developing new targeting compounds that can be used to selectively modulate the functions of HMPs to obtain a biological activity of interest (e.g. new drugs with reduced toxicity and side effects) has been severely hampered by the lack of 3D structures for nearly all human HMPs.
This lack of 3D structures of HMP has motivated extensive efforts and huge levels of funding around the world to determine the structures using protein crystallography, but currently there are accurate experimental structures for only about 120 HMPs and for only one human 7TM protein of the 800. The first experimental 3D structure for any membrane protein (MP) was published in 1985 [4] for the photosynthetic reaction center. Since then experimental structures have been obtained for only about 160 unique MPs, of which only 10 are human. For 7TM proteins (a particularly challenging class of MPs) of any form of life, the first structure was determined in 2000 for bovine rhodopsin [5], the second structure was determined in 2007 for a modified human β2 adrenergic receptor [6], and the third structure was determined in 2008 for squid rhodopsin [7]. Thus of the 800 human 7TM structures needed for drug optimization, just one structure is known experimentally and obtaining all others is destined to require decades and huge additional funding.
Moreover, since crystallographic observation requires that all molecules in the crystal have the same conformation, the few 7TM structures observed so far have been modified to prohibit flexibility and/or binding a ligand that locks in a specific conformation. This is a significant limitation of the crystallographic approach, since it is well established that the process of activating a 7TM protein involves a sequence of conformational changes that enable the cell to convert extracellular signals into physiological responses [8].
Current experimental methods provide not even the promise of ever enabling resolution of the dynamical structures through which the 7TM protein evolves during activation. This provides an even more significant limitation in the experimental study of the 7TM proteins since it is expected that different types of 7TM ligands, such as agonists, antagonists, inverse agonists, and modulators may bind to different receptor conformations, and that some 7TM proteins may act as dimers or multimers.
An alternative to experimental determination of 3D structures is the use of homology computational methods. Here one aligns the amino acid sequence of the unknown with that of one or more known structures, accounting for various non-alignable parts (gaps and bulges) to build the structure for the unknown using the known as a guide. To obtain a sufficiently accurate structure for ligand design (where ligand can be a drug) aimed at altering the function of HMPs, homology computational methods generally require comparison to a number of related experimental structures, ideally with a sequence identity well over 40%. In contrast, there are only three structures for 7TMs and the sequence identity desirable to provide predicted structures suitable for applications such as drug design is significantly higher than the sequence identify detectable by this approach alone. In a further approach illustrated by PCT patent publication WO/2005/17805, incorporated herein by reference in its entirety, a “MembStruk” method is used for predicting a single low-energy structure for a multipass TM protein.